Modern Creatinine (Bio)Sensing Challenges of Point-Of-Care Platforms

Biosensors and Bioelectronics 130 (2019) 110–124

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Biosensors and Bioelectronics

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Modern creatinine (Bio)sensing: Challenges of point-of-care platforms T ⁎ ⁎ Rocío Cánovas, María Cuartero , Gastón A. Crespo

Department of Chemistry, School of Engineering Science in Chemistry, Biochemistry and Health, Royal Institute of Technology, KTH, Teknikringen 30, SE-100 44 Stockholm, Sweden

ARTICLE INFO ABSTRACT

Keywords: The importance of knowing creatinine levels in the human body is related to the possible association with renal, Creatinine muscular and thyroid dysfunction. Thus, the accurate detection of creatinine may indirectly provide information Early diagnosis surrounding those functional processes, therefore contributing to the management of the health status of the Healthcare individual and early diagnosis of acute diseases. The questions at this point are: to what extent is creatinine POC devices information clinically relevant?; and do modern creatinine (bio)sensing strategies fulfil the real needs of Enzymatic biosensors healthcare applications? The present review addresses these questions by means of a deep analysis of the Blood analysis creatinine sensors reported in the literature over the last five years. There is a wide range of techniques for detecting creatinine, most of them based on optical readouts (20 of the 33 papers collected in this review). However, the use of electrochemical techniques (13 of the 33 papers) is recently emerging in alignment with the search for a definitive and trustworthy creatinine detection at the point-of-care level. In this sense, biosensors (7 of the 33 papers) are being established as the most promising alternative over the years. While creatinine levels in the blood seem to provide better information about patient status, none of the reported sensors display adequate selectivity in such a complex matrix. In contrast, the analysis of other types of biological samples (e.g., saliva and urine) seems to be more viable in terms of simplicity, cross-selectivity and (bio)fouling, besides the fact that its extraction does not disturb individual's well-being. Consequently, simple tests may likely be used for the initial check of the individual in routine analysis, and then, more accurate blood detection of creatinine could be necessary to provide a more genuine diagnosis and/or support the corresponding decision-making by the physician. Herein, we provide a critical discussion of the advantages of current methods of (bio)sensing of creatinine, as well as an overview of the drawbacks that impede their definitive point-of-care establishment.

1. Introduction the muscles and other parts of the body, CRE is transported through the bloodstream by the kidneys and excreted in the urine. Accordingly, Creatine is mainly synthesized in the kidneys, liver and pancreas both fluids (blood and urine) deserve clinical attention with respect to (Fig. 1a) before being transported to the tissues and organs, where it is CRE levels (Killard and Smyth, 2000). metabolized to creatinine (Fig. 1b), termed CRE. When adenosine tri- CRE is the second most analysed biomolecule for clinical purposes phosphate is involved in this conversion, creatine per se is a source of after glucose (Joffe et al., ).2010 Thus, CRE values outside of typical energy for many biological processes, such as muscle activity ranges is evidence of any health issue associated with renal, muscular (Narayanan and Appleton, 1980). In this context, the creatine to CRE and thyroidal functioning; and levels fairly beyond healthy ones en- cyclization rate is not fully understood despite this knowledge poten- compass very serious diseases, such as chronic kidney disease (CKD), tially assisting in the control of degenerative diseases of the muscles as different types of muscular disorders, cardiovascular problems oreven well as improvement of sport performance (Diamond, 2005). In con- Parkinson's disease, among others. As an example, when CRE levels in trast, it is well-known that CRE levels are fairly constant in the human the serum (i.e., blood) are below 40 µM (Table 1), this indicates an body, mainly depending on the muscle mass of the individual abnormal reduction in muscle mass (Killard and Smyth, 2000). How- (Narayanan and Appleton, 1980; Pundir et al., 2013). For example, the ever, for concentrations greater than 150 µM (Table 1), additional typical reference ranges for serum levels of CRE in healthy patients are analytical tests are required to exclude any risk of CKD. In extreme on the order of 45–90 μM for women and 60–110 μM for men (see cases, values above 500 µM inform of a clear renal impairment that will Table 1; Randviir and Banks, 2013). After subsequent CRE generation in likely involve dialysis treatment or kidney transplantation (Killard and

⁎ Corresponding authors. E-mail addresses: [email protected] (M. Cuartero), [email protected] (G.A. Crespo). https://doi.org/10.1016/j.bios.2019.01.048 Received 18 November 2018; Received in revised form 11 January 2019; Accepted 20 January 2019 Available online 30 January 2019 0956-5663/ © 2019 Elsevier B.V. All rights reserved. R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124

Fig. 1. General scheme of the metabolism of Creatine to CRE. Creatine ori- ginated in different organs is metabolized into CRE in muscles and brain. a) Illustration of the synthetic route of Creatine by the organism together with the main organs in which these processes take place (kidneys, liver and pancreas). AGAT: L-arginine: glycine amidinotransferase and GAMT: S-ade- nosyl-L-methionine: N-guanidinoacetate methyltransferase. Black arrows show where Creatine is required as a source of energy (producing CRE), mainly brain and muscles. b) Creatine metabolization to CRE through hydrolysis reactions or involving phospho- creatine as an intermediate. CK: Creatine kinase; biosynthesis of creatinine occurs during the reaction.

Table 1 serum measurements, as well as the albumin-to-CRE ratio in urine to Ranges of CRE levels found in different matrices from healthy and unhealthy evaluate the state of kidney activity (Cockcroft and Gault, 1976; Junge individuals. et al., 2004; Levey et al., 2015; Omoruyi et al., 2012; Tziakas et al., Sample pH rangea Healthy levels Harmful levels [CRE]a 2015). The early diagnosis of any sort of dysfunction is extremely im- [CRE] portant in the prevention and/or control of CKD because this illness is asymptomatic during the first stages (mild and moderate CKD). Hence, b c c Urine 4.5 – 8 4.4 −18 mM < 3 and > 20 mM when the individual notices any symptom, the disease is likely very Serum / Blood 7.35–7.45 45 – 90 µM (♂) < 40 µM and > 150 µM 60 – 110 µM (♀)d (up to 1 mM)d advanced, and the kidneys are commonly quite damaged. Altogether, Saliva 6.2–7.4 4.4–17.7 µMe 17.7 up to 591 µMe the patient experiences a radical change in their life because CKD Sweat 4–6.8 9.4–18 µMf 60 – 200 µMf treatment involves visiting the hospital every two days for dialysis or, if Tears 6.5–7.5 – – the damage is too severe, that person would need a kidney transplant. ISF 7.2–7.4 g g Importantly, over one million people worldwide are undergoing dia- a (Corrie et al., 2015). lysis treatment. Moreover, the incidence of renal failure has doubled b The range of pH can vary also depending on the different laboratories and, over the last 15 years (Bagalad et al., 2017). Therefore, there is a so- in the case of urine, it can be even wider in case of infection. cietal need for trustable detection of CRE at the point-of-care (POC) c (Li et al., 2015; Ruedas-Rama and Hall, 2010). level not only to allow one to preserve individual well-being and con- d (Killard and Smyth, 2000; Tseng et al., 2018). tribute to a more affordable healthcare system, but also to monitor e (Lloyd et al., 1996; Venkatapathy et al., 2014). patients at advanced stages of CKD, which necessitates more than one f (Al-Tamer et al., 1997; Bass and Dobalian, 1953). The symbol ♂ indicates analysis per day during dialysis treatment (Hannan et al., 2014; males and ♀ in females. Michalec et al., 2016). g Similar levels of CRE can be found in ISF and serum (Ebah, 2012; CRE detection is also crucial in the premature diagnosis of various Kayashima et al., 1992). muscular diseases, such as Duchenne muscular dystrophy (Fitch and Sinton, 1964), myasthenia gravis, acute myocardial infarction Smyth, 2000). Furthermore, persistently elevated levels of CRE may (Radomska et al., 2004b) or guanidinoacetate methyltransferase reveal a high risk of mortality (Levey et al., 2015). Notably, the inter- (GAMT) deficiency (Diamond, 2005), as well as verifying patient status pretation of CRE observations must be always carried out considering before and after surgical interventions (Ho et al., 2012; Prowle et al., the muscle mass of the patient as the same value may be considered 2014; Spahillari et al., 2012; Vart, 2015) or when suffering an accident, normal in a young male with a relatively high muscle mass or may as muscular lesions are somewhat related to higher concentrations of indicate CKD in elderly females (Tseng et al., 2018). CRE in the bloodstream. Besides this, CRE detection is valuable for There are a number of organizations, such as the American verifying dehydration status in individuals, e.g., as a consequence of a Association of Kidney Patients, American Kidney Foundation, National decrease of renal blood flow during or after engaging in strong physical Kidney Foundation in New York and the Nephron Information Center, activity (Baxmann et al., 2008; Mohamadzadeh et al., 2016). In addi- among others1 that define five different stages for kidney function tion, the administration of certain drugs and/or treatments, such as based on the estimated glomerular filtration rate (eGFR), which in turn acetylcholine inhibitors, cyclosporin or chemotherapy, may present is calculated from CRE levels in serum together with other factors, such side-effect damage in the kidneys, and therefore, high levels ofCREare as gender and age. In addition, CRE clearance is computed from CRE probably observed owing to elevated renal impairment (Cherney et al., 2017; Kulling et al., 1995; Wiebe et al., 2017). In all these cases, POC 1 detection of CRE should supply a real-time analysis that yields clinical www.aakp.org; www.kidneyfund.org; www.kidney.org; www.nephron.com. information regarding renal functionality before, during and after

111 R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124 therapy or physical activity. when picric acid interacts with CRE to form a chromogen that absorbs Despite the proven necessity of CRE detection at the POC level as within the wavelength range of 470–550 nm (red colour) with a normal mentioned before, currently, analysis is always carried out in cen- maximum at ca. 520 nm (Mohabbati-Kalejahi et al., 2012). Despite this tralized laboratories after extracting the sample. In contrast, the defi- reaction being nonspecific for CRE, in fact, the method's sensitivity is nitive clinical analysis of CRE (at the POC level) must provide the affected by temperature, pH and others variables (Walsh and Dempsey, features proposed in the ASSURED guidelines (St John and Price, 2014): 2002); the Jaffé method is still widely used owing to the rapid analysis the detection has to be Affordable, Sensitive (minimal false negatives), (~15 min per sample), its cost-effectiveness and the fact that itis Specific (minimal false positives), User-friendly (simple enough tobe adaptable for automatization (Narayanan and Appleton, 1980; Spierto conducted by the patient in remote settings), Rapid and Robust et al., 1979). Importantly, CRE concentrations determined by this (avoiding hospital wait times and providing reliability), Equipment-free method must be carefully interpreted because of a number of reasons: (or minimal equipment needed) and easily Delivered (to the end user) (Wang et al., 2016; Zarei, 2017). The present review critically analyses (i) The reaction is extremely sensitive to temperature, e.g., a variation the reported (bio)sensors from 2014 as alternative approaches for the of 1 °C produces an error of ± 0.7 µM in the estimated CRE con- current analysis of CRE carried out in hospitals and clinical labora- centration, which can lead to erroneous decision-making in cases tories. For this purpose, it is important to establish in which biological of a potential decrease in muscle mass (Narayanan and Appleton, samples CRE is present and what kind of clinical information is to de- 1980; Spierto et al., 1979). note its detection in each particular fluid. (ii) The risk of sample contamination is relatively high in the case of Previous reports on CRE detection have collected papers published blood analysis as colorimetric detection cannot be performed di- up to 2013 mainly describing CRE sensing concepts that were essen- rectly in blood because of two main reasons. First, the red colour of tially classified into optical methods and biosensors based on enzymatic the blood impedes the detection of the chromogen adduct in the reactions (Lad et al., 2008; Killard and Smith, 2000; Shephard, 2011; red band. Second, proteins and other compounds strongly interfere Mohabbati-Kalejahi et al., 2012; Randviir and Banks, 2013; Pundir in the derivation reaction. Consequently, the technique is applied et al., 2013). Nevertheless, while CRE biosensing was claimed as the to serum and not whole blood (Doolan et al., 1962; Owen et al., most promising alternative to substitute the well-established Jaffé 1954). method, other techniques also showed analytical features suitable for (iii) Spectrophotometric analysis generally requires a sample volume the clinical analysis of CRE. In this sense, Lad et al. (2008) reported on from 50 µL to 2 mL depending on the instrumentation available in analytical characteristics and several designs of electrochemical bio- the laboratory (Walsh and Dempsey, 2002). Indeed, certain spec- sensors based on different recognition elements such as enzymes, an- trophotometers operate with volumes of just a few microliters (0.5 tibodies and molecular imprinted polymers (MIPs). Killard and Smith – 5 µL).2 There are also commercially available kits with the re- discussed on the advantages and limitations of potentiometric and quired amount of picric acid for specific sample volume3. In other amperometric biosensors based on enzymatic CRE recognition pub- cases, a sample dilution is normally needed to provide a CRE lished up to 2000. Shephard (2011) reviewed all the POC devices concentration within the linear range of response of the calibra- available for measuring CRE in whole blood, serum and plasma up to tion. For instance, in the case of urine, a 5 – 20 dilution factor is 2011. The review by Mohabbati-Kalejahi et al. (2012) highlighted that traditionally demanded while even larger dilutions (400–800 fold) chromatographic CRE analysis provided improved limit of detection (in could be needed.3 During serum analysis, this is less common. the order of 0.28 nM) while potentiometric electrodes presented fast However, the total volume of blood or urine collected from the response time and those based on MIPs displayed the best selectivity. individual depends on the final aim and type of whole analysis, Randviir and Banks (2013) summarized the analytical methodologies i.e., the nature and range of parameters to be analysed in the same (enzymatic and non-enzymatic) utilized to quantify CRE up to 2013. sample. In the case of urine, as the CRE levels can fluctuate de- Pundir et al. (2013) published a work focused on the status of enzy- pending on diet, hydration and other factors (Boeniger et al., matic and non-enzymatic electrochemical CRE detection as well as 1993), a single spot check is insufficient for decision-making and certain immunosensors and the introduction of nanomaterials towards therefore, the average content in urine collected over 24 h is pre- the development of smart sensing devices. Beyond providing an up- ferable in terms of an accurate clinical indication. Overall, the dated collection of published papers on CRE (bio)detection from 2014 required blood and urine collection as well as manipulation is up to the present, this review ‘puts on the table’ for the first time the considered tedious and risky, and the dilution requirement will advantages of modern CRE (bio)sensing together with the analytical inherently involve an error in CRE concentration estimation drawbacks that impede definitive POC establishment. (Narayanan and Appleton, 1980). (iv) Regarding CRE levels in urine, this is normally expressed as CRE 2. In which human fluids is it plausible to determine creatinine to clearance by kidneys, which can be estimated following two ap- obtain clinically relevant information? proaches (Doolan et al., 1962). One is based on blood measure-

ments ([CRE]serum = CRE mg in 100 mL of blood) and employs the CRE is present in a variety of biological fluids (Table 1) because of Cockcroft-Gault formula that considers a fixed value for patient's its participation in diverse metabolic routes, as already described in the gender (72 for males and 85 for females; in the latter, the number introduction (Narayanan and Appleton, 1980). As a result, the analysis 72 in Eq. (1) has to be replaced), weight and age (Eq. (1)) of CRE in distinct samples provides different types of clinically relevant (Cockcroft and Gault, 1976). information. For example, CRE detection is already included in routine (140age ) × weight blood analysis as a preliminary check for the malfunction of kidneys. CREclearance = 72[CRE ]serum (1) Typically, CRE detection is accomplished in clinical laboratories after blood extraction. Note that centralized measurements are the opposite The second approach consists of a comparison between the average to the ASSURED requirements to establish a POC diagnosis platform (St CRE levels excreted in 24-h urine by the individual and the blood John and Price, 2014). On the other hand, CRE analysis in urine is also content after this collection time (Mohabbati-Kalejahi et al., 2012; very common, which is performed during a 24 h sample collection to provide more accurate results (see more details subsequently). Both blood and urine analysis are based on the Jaffé method (Greenberg 2 www.metrixlab.mx et al., 2012). 3 https://www.alpco.com, www.mybiosource.com, www.abcam.com, The Jaffé method involves a colorimetric readout of the sample www.sigmaaldrich.com

112 R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124

Narayanan and Appleton, 1980; Schwartz et al., 1987). In prin- Saliva is generally a robust estimator of what takes place in the ciple, the latter should provide more accurate results because it blood. There are a variety of well-known correlations in terms of re- considers the CRE content in two different biological fluids rather levant parameters extracted from saliva that may take the place of than an empirical formula (Cockcroft and Gault, 1976). analysing them directly in blood, e.g., proteins, biomarkers and in- (v) While the Jaffé method is considered the gold standard for CRE gested drugs, among others (de Almeida et al., 2008; Corrie et al., 2015; detection, it normally provides CRE levels slightly higher than Lee and Wong, 2009; Maccallum and Austin, 2000; Pfaffe et al., 2011). other analytical techniques (ca. 3–5%). Indeed, extreme differ- Indeed, saliva has been demonstrated as an alternative to monitor ences (even greater than 10%) were reported for some isolated metabolic by-products of kidney failure (Lloyd et al., 1996; Nagler, cases involving HPLC and amperometric techniques (Walsh and 2008). For example, CKD is one of the systemic diseases that can affect Dempsey, 2002). This fact has to be considered as another warning the contents of salivary secretions (Lasisi et al., 2016). It has been re- for the careful clinical interpretation of analytical observations. ported that CRE enters saliva via ultrafiltration, and its salivary con- Besides, it is important to consider the toxicity and explosive issues centration seems to range from 10% to 15% of that found in blood of picric acid, which additionally require expertise by the end user (Lloyd et al., 1996). Whereas studies have shown promising results (He et al., 2015). based on the comparison of CRE levels detected in saliva and serum (vi) Biological samples have different pH values (Table 1) and this has samples, for instance, accuracy in the range of 90–95% (Bagalad et al., to be considered when applying the Jaffé method for comparative 2017; Venkatapathy et al., 2014), saliva analysis has less diagnostic purposes as the ratio of alkaline picrate and alkaline CRE picrate accuracy and specificity versus traditional colorimetric detection of varies with pH, therefore influencing the final colour of the sample CRE in serum (Xia et al., 2012; Lasisi et al., 2016). However, these (Narayanan and Appleton, 1980; Owen et al., 1954). results could be influenced by the inherent error associated with the employment of the Jaffé method as well as all the steps involved in It is worth mentioning that there is additional clinical information sample collection and storage (Bagalad et al., 2017). that can be drawn from blood and urine analysis, such as the urea ni- Even though the use of saliva in diagnostic tests has many ad- trogen-to-creatinine ratio in blood as well as the potassium-to-creati- vantages (i.e., non-invasive and affordable collection in terms of com- nine and albumin-to-creatinine ratios in urine (BUN-CRE, K-CRE and A- plexity and costs, appropriateness for all age groups, convenience for CRE ratios, respectively) (Bartz et al., 2015; Lin et al., 2009). Once the use in non-laboratory areas and the screening of large population BUN-CRE ratio is higher than normal (from 10:1–20:1), this may in- (Pfaffe et al., 2011), suitability for patients suffering from clotting dicate the malfunction of the kidneys or presence of stones or tumours disorders and with compromised venous access (Gröschl, 2008; Lee and (Winarta et al., 2015). Moreover, once the BUN-CRE ratio is too high, Wong, 2009; Nagler, 2008; Venkatapathy et al., 2014)), it is important severe conditions consisting of kidney failure, shock or hydration can to remark that salivary concentrations of certain compounds can be arise (Lin et al., 2017). Conversely, when the ratio is too low, this can affected by many factors, not only including gender, but also dietand be caused by malnutrition, poor diet in terms of protein or severe liver genetics (Fahed et al., 2012; Liu et al., 2013). As a result, the utility of damage (Winarta et al., 2015). saliva as a general diagnostic fluid is still subject to continuous em- The simultaneous analysis of CRE together with potassium or al- pirical evaluation (Lasisi et al., 2016). While serving as a promising bumin in urine collected for 24 h or as one-spot may also have clinical candidate for POC detection of CRE, there is plenty of room for in- usefulness. For example, if the K-CRE ratio is greater than 1.5, this is vestigation in this direction, especially considering the accuracy of the indicative of renal potassium waste because of any organism dysfunc- results, noting that only 18 of the 33 papers presented in Table 2 have tion (Jȩdrusik et al., 2017). In this context, Assadi et al. reported a been validated with a standard method, and their translation into valuable study based on specific clinical cases, showing what type of trustworthy clinical decision-making. diseases can be caused by various metabolic conditions together with Another alternative is the use of sweat, which is being accounted for the K-CRE ratio (Assadi, 2008). In addition, different organizations in the development of many wearable sensors at present (Bandodkar (such as the American Diabetes Association, International Diabetes et al., 2016; Florea and Diamond, 2015; Parrilla et al., 2018a). Col- Federation, National Kidney Foundation, Caring for Australians with lection of sweat is also non-invasive, but typically provides con- Renal Impairment, UK Renal Association Clinical Practice Guidelines siderably less sample volume than saliva. Moreover, there is one in- for the Care of Patients with CKD, among others) recommend the utility convenience associated with the procedure of collecting samples based of the A-CRE measurement when a 24-h urine collection period is not on exposing the patient to high temperatures to facilitate an enhanced available. The concentration considered the ‘decision limit’ reported by sweating rate. For example, when patients are elderly, they may ex- laboratories varies from 15 to 30 mg/L for urine albumin and perience side effects related to exposure to high temperatures, such as 1.0–3.6 mg/mmol (9–32 mg/g) for A-CRE (Miller et al., 2009). Risk for low blood pressure or dizziness. In addition, an inevitable evaporation both cardiovascular and kidney disease rises significantly with urine during the collection is generally translated into overestimation of CRE ACR above 10 mg/g (Kramer et al., 2017). Moreover, A-CRE can be levels (Al-Tamer et al., 1997). valuable for predicting acute kidney injury during hospitalization for Eccrine sweat fluid has been shown to contain several biochemical acute myocardial infarction (Tziakas et al., 2015), cardiovascular markers of clinical interest (Sato et al., 1989), and comprises the nat- events and diseases in adults (Bartz et al., 2015; Cho et al., 2015), as ural route for the excretion of biological waste products, as is the case well as early detection of CKD (Omoruyi et al., 2012). Nonetheless, A- when kidneys are damaged and their function compromised (Al-Tamer CRE varies depending on the age, gender and ethnicity, and therefore, et al., 1997). Nonetheless, early evidence has suggested that CRE con- medical decision making should be carefully undertaken (Marcovecchio tent in sweat is closely linked to the rate of sweating rather than with et al., 2018). the level of CRE in blood (Ladell, 1947). Hence, the slower the rate of One advantage of using urine versus blood is the easy sample col- sweating, the higher the CRE content. Additional studies have revealed lection that generally confers no disturbance to patient integrity, unlike how the rise in CRE content in the blood is correlated with an increase with needle-based extractions. However, overall and in spite its com- of CRE in saliva, but varies slightly with respect to sweat content. This plexity, blood offers the most rapid and clinically relevant detection of fact may likely impede the selection of sweat as a fluid for CRE de- CRE (Peake and Whiting, 2006). Nevertheless, the use of other bio- tection, otherwise at least a constant sweating rate is achieved by all matrices is gaining more and more interest with respect to non-invasive individuals, and therefore, all results may be standardized. analysis beyond urine. However, this trend has not yet been outlined in Another biological fluid considered for CRE detection is tears. (bio)sensors reported over the last five years for CRE detection (see Despite the high variety of compounds present in this fluid (electro- Table 2 and the next section). lytes, organic salts, proteins, glucose, urea and other biopolymers)

113 .Cnvse al. et Cánovas R. Table 2 Summary of the CRE (bio)sensors reported in the literature from 2014 up to now.

Readout Description Sensing principle Working range LOD Analysis Time Validation Sample POC Ref.

Colorimetry Paper-based chip with CMOS camera Reaction with picric acid / NaOH 17.7 – 707 µM – 5 min Correlation R2 Serum Noa Fu et al. (2018) and USB connection (Jaffé) = 0.9994 Colorimetry µPAD with CMOS camera and Wifi Reaction with picric acid / NaOH 16.8 – 675 µM 16.8 µM 8 min Correlation R2 Whole blood Yes Tseng et al. (2018) chip (Jaffé) = 0.9920 Colorimetry AgNPs-based sensor Alkaline CRE aggregation with citrate- 0 – 4.2 µM 53.4 nM 1 up to 10 min – Diluted urine No Alula et al. (2018) capped AgNPs Colorimetry Photosensor based on LEDs and LCD Enzyme cascade (BIOSENSOR) 67.2 – 60 µM 5 min – Plasma Yes Dal Dosso et al. (2017) screen 1768 µM Colorimetry AuNPs-based sensor Synergistic adenosine/CRE – 12.7 nM < 5 min – Artificial urine No Du et al. (2016) coordination Bovine serum Colorimetry AgNPs-based sensor AgNPs coated with picric acid (Jaffé) 0.01 – 1 µM 8.4 nM 4min – Serum CSF No Parmar et al. (2016) Colorimetry Paper-based strips Enzyme cascade (BIOSENSOR) 0.22 – 0.17 mM 11 min Correlation R2 = 0.977 Urine No Talalak et al. (2015) 2.21 mM Colorimetry CDS with camera and CSPT array Jaffé 160 µM – 89 µM 5 – 15 min Correlation R2 = 0.930 Diluted urine No Debus et al. (2015) 1.6 mM – 0.977 Colorimetry AuNPs in solution Cross-linking reaction 0.1 – 20 mM 80 µM 24 min – Diluted urine No He et al. (2015) Colorimetry plasmonic nanoparticles CRE with uric acid and AuNPs 0.1 – 0.4 mM 19.8 nM > 5 min Recovery 94 – 103% Diluted urine No Du et al. (2015) modified with Hg2+ Colorimetry Label-free AuNPs system SPE of CRE with AuNPs 0.13 – 0.12 mM 10 – 30 min – Diluted urine No Sittiwong and Unob 0.35 mM (2015) Absorbance / Chemodosimeter PTP chalcone 0.1 nM – 0.058 nM 10 min Recovery 88 – 105% Serum No Ellairaja et al. (2018) Fluorescence 1.4 mM Optical (Liquid crystals) DMOAP-coated glass slides Hydrolysis of CRE with deprotonation – as low as 3 – 30 minb – Artificial samples No Verma et al. (2016) of HBA (change in orientation of LCs) 50 µM 114 Fluorescence (light-up) Pd2+ naphthalimide-based Synthetic route leading to a – as low as 30 min Recovery 5 – 99% Serum No Pal et al. (2016) fluorescence probe degradation complex 0.30 µM ECL ITO electrode with NiNCs Magnetic graphene oxide (GO-Fe3O4) 5 nM – 1 mM 5 nM 15 – 30 min Recovery 93 – 100% Diluted serum No Babamiri et al. (2018) in MIP Diluted urine PL carbon nanodots BSA + CRE 17 µM – 6.2 µM 5 min – Artificial samples No Babu et al. (2018) 1.7 mM 2+ CL Co -based system CRE + H2O2 0.1 – 30 µM 72 nM 1 min Recovery 100 – 103% Diluted urine No Hanif et al. (2016) SPR gold grating substrate AgNPs deposited with PDADMAC and 0.1 – 20 mM – > 10 min – Artificial samples No Pothipor et al. (2017) PSS S-SERS NPGD plasmonic substrates CRE interaction with NPGD 100 nM – 13.2 nM (in Ca. 1 h – Artificial samples No Li et al. (2015) 100 μM water) IMS PPy/GO fibres SPME with fast evaporation 0.01 – 4.4 mM 5.3 µM 7 – 10 min Recovery 92 – 104% Spiked diluted No Jafari et al. (2015) 0.02 – 2.2 mM 22.9 µM and 101 – 110% urine and plasma CV Carbon ink screen printed electrodes Complex formation with cooper 6 – 378 µM 0.07 µM > 30 min Recovery 95 – 98% Serum No Raveendran et al. (2017) 2 Biosensors andBioelectronics130(2019)110–124 CV PINE electrodes coated with FeCl3 FeIII binding 0.88 – 22 mM – < 1 min Correlation R = 0.91 Urine Yes Kumar et al. (2017b) cotton membranes CV and DPV Graphite electrode CdSe semiconductor QDs 0.44 µM – 0.23 µM Ca. 2 h – Urine Serum No Hooshmand and 8.84 mM Es'haghi (2017) CV and DPV MWCNT screen printed electrode Enzymatic recognition by papain – 0.006 nM 10 min – Spiked urine No Desai et al. (2018) (cysteine protease) (BIOSENSOR) DPV GC electrode with Ni-PANI NPs Magnetic molecularly imprinted 0.004 – 0.8 µM 0.2 nM Ca. 3 h Recovery 95 – 102% Artificial sample No Rao et al. (2017) polymer DPV Screen printed gold electrode Molecularly imprinted polymers 0.88 – 8.84 nM 0.14 nM 2 – 5 h Correlation R2 = 0.99 Diluted urine No Diouf et al. (2017) (MIPs) DPV Magnetic GC electrode with Fe3O4- Magnetic molecularly imprinted 0.02 – 1 µM 0.35 nM Ca. 55 min Recovery 90 – 105% Artificial samples No Wen et al. (2014) PANI NPs polymer Amperometry PINE electrode FeCl3 electroactivity 17.6 – – > 10 min – Saline media No Dasgupta et al. (2018) 512.7 µM (continued on next page) R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124

(Zapata et al., 2005), large-sized biomolecules, like CRE, are found in very low concentrations likely because of solubility issues (Gaebler and Keltch, 1927; Sivapragasam et al., 2016). Some decades ago, Kang et al. (1988) presented the different contents of CRE found in blood and tears from 30 healthy control patients (mean values of 83 versus 80 µM, respectively) and 10 end-stage CKD patients, the latter in which CRE Guinovart et al., (2016, 2017) Braiek et al. (2018) Kumar et al. (2017a) Zhybak et al. (2016) Ozansoy et al. (2017) amounts in tears from pre- and post-haemodialysis treatment were compared. Interestingly, following haemodialysis treatment, CRE con- No POC Ref. tent decreased 36% in the case of tears and 48% in blood. Later, Zapata et al. (2005) confirmed the same in horses, where the mean concentration of CRE in tears was 9.6% lower than in serum. Overall, there is lack of information regarding CRE in tears, both healthy and harmful levels, and the correlation with blood content. Cerebrospinal fluid (CSF) is another fluid that could be potentially Diluted plasma Artificial serum No Artificial sample No Sample considered for CRE detection. However, sample extraction involves a high level of intrusion in patients. Consequently, the use of CSF is not discussed in this section as it is not compatible with the POC concept. In = 0.99 Diluted serum= 0.99 Diluted serum No No = 0.966 Diluted urine 2 2 2 contrast, CRE is also found in interstitial fluid (ISF) (Kost et al., 2000; Paliwal et al., 2013), which has recently been the target of certain wearable sensors that were created with the goal of composition characterization (Parrilla et al., 2018b). Importantly, small differences Correlation R Correlation R Correlation R are found in CRE concentrations between plasma and ISF in healthy patients. Indeed, individual ISF concentrations are strongly correlated with plasma CRE (Pearson´s correlation coefficient of 0.94) (Ebah, 2012), which was additionally confirmed in the case of rabbits > 3 min – 1 min 1 – 4 min – Analysis Time Validation (Kayashima et al., 1992). However, it seems that dialysis treatment could increase this difference (Ebah, 2012). Kost et al. (2000) showed this fact by means of an in vivo comparison in rats after provoking a e faster CRE extraction through ultrasound. Unfortunately, as in the case of tears, there is a lack of accessible information regarding CRE in CSF. (urine)

3. Modern sensing of creatinine

The very first CRE sensor, apart from the Jaffé method, was reported by Meyerhoff’s group in 1976 and it consisted of a gas-sensing ammonia 0.3 – 0.6 mM 1 µM – 10 mM 0.63 µM Working range LOD electrode (Meyerhoff and Rechnitz, 1976). The electrode partially used the natural monoenzymatic pathway by which CRE is hydrolysed with ammonium generation: + A. 1 : Creatinine+ H2 O N methyl hydantoine+ NH4

+ + A. 2 : NH4 + oxoglutarate+ NAD() P H Glutamate+ NAD() P + H

The required enzymes are creatinine deaminase (CD, EC 3.5.4.21) (Step A.1) and glutamate dehydrogenase (GDH, EC 1.4.1.3) (Step A.2). This sensor has served as source of inspiration for subsequent studies based on the indirect detection of CRE by ammonium sensing Enzymatic recognition (BIOSENSOR) 10 – 600 µM 2 µM cations with ionophore Enzymatic reaction (BIOSENSOR) 1 – 125 µM 0.5 µM 15 s Sensing principle Enzyme cascade (BIOSENSOR) 0.01 – 12 µM 0.01 µM 2 s through various methods (Magalhães and Machado, 2002; Mascini and

= graphene oxide – iron oxide nanoparticles; NiNCs = nickel nanoclusters; MIP = molecularly imprinted polymer; PL = photoluminescence; BSA = bovine Palleschi, 1982). In addition, pH detection (Zhybak et al., 2016) and the 4 O

3 electrochemical monitoring of the oxidation of NADH were employed for indirect CRE detection on the basis of this reaction (Fossati et al., 1994). Indeed, there has been a general trend over the years to derivatize CRE to another compound measurable primarily through colorimetry or electrochemistry (Killard and Smyth, 2000; Lad et al., 2008; Mohabbati-Kalejahi et al., 2012; Pundir et al., 2013; Randviir et al., 2013). Table 2 lists papers focusing on CRE detection that have been published since 2014 to date. A preliminary assessment reveals that the Interdigitated electrode with contact printing based enzymatic membrane GC electrode with selective membrane Selective recognition of creatininium Zeolites based electrodes and AuNPs Enzymatic reaction (BIOSENSOR) Up to 2 mM 5–10 µM SPE with Cu, Nafion, PANIenzyme and the Description Enzyme NPs immobilized in GC electrode use of the Jaffé method is being replaced by other colorimetric approaches as well as electrochemical (bio)sensors. Additional techniques, such as (electro or photo)chemical luminescence (ECL, PL and CL), surface plasma resonance (SPR) and mass spectrometry (MS) have also been proposed in the literature. However, the large and sophisticated instrumentation involved in these approaches is not compatible ( continued ) with POC detection of CRE. Moreover, only colorimetric and voltam- This time can increase up to 3 h depending on the enzyme concentration as well as the CRE concentration. “These human serum creatinine samples are obtained from whole blood samples via a separation and purification process at the hospital”.

a b metric sensors have been applied in a truly POC manner (three of 33 Conductometry Potentiometry ISFET Amperometry Readout Amperometry serum albumin; CL = chemiluminescence;Raman SPR scattering; NPGD = surface = nanoporous plasmonvoltammetry; gold resonance; QDs disk; PDADMAC = IMS quantum = = poly dots; ion MWCNT (diallydimethylammonium mobility = chloride); spectrometry; multiwalled PPy/GO PSS carbon = = nanotube; Polypyrrole/graphene poly GC oxide; (sodium SPME = 4-styrenesulfonate); glassy = Solid S-SERS carbon; phase = PANI stamping microextraction; = polyaniline; surface CV ISFET = enhanced cyclic = Ion voltammetry; DPV Sensitive = Field differential Effect pulse Transistor. Table 2 CMOS = complementary metal oxide semiconductor;= USB liquid = crystal universal display; serial AuNPs bus; =(pyren-2-yl)− µPAD gold 1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one; = nanoparticles; microfluidic CSF DMOAC paper-based = analytical cerebrospinal = N,N-dimethyl-N-octade-cyl-3-aminopropyltrimethoxysilyl device; fluid;trochemiluminescence; CDS chloride; ITOAgNPs = = circular = HBA Indium-tin silver nanoparticles; dichroism spectroscope; = oxide; Hexylbiphenyl-4-carboxylic GO-Fe CRE = creatinine; acid; CSPT = computer LCs LEDs = light-emitting screen photo-assisted = liquid diodes; LCD technique; crystals; SPE ECL = solid phase = elec- extraction; PTP = (E)− 3- papers) (Dal Dosso et al., 2017; Kumar et al., 2017; Tseng et al., 2018),

115 R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124 but without achievement of implementation in real scenarios as accu- that the physiological pH of the sample must be modified and main- rate platforms yet. tained at pH 12 to force the aggregation. The urgent need for decentralizing CRE analytical detection makes Du et al. (2016) proposed a AuNPs-based sensor that takes ad- it more evident that chromatographic techniques are not appropriate to vantage of the synergistic coordination of adenosine and CRE in the resolve this analytical challenge despite there being remarkable success presence of silver ions. Despite measurements in artificial urine and in the analysis of the majority of biological fluids at the laboratory scale bovine serum being successfully carried out, the validation of the sensor 15 years ago (Kochansky and Strein, 2000; Walsh and Dempsey, 2002). was somehow missed. In addition, He et al. (2015) also made use of a In principle, the selected technique as a readout for the (bio)sensing of sensing concept based on AuNPs, but the detection principle consists of CRE at the POC level will be a compromised situation in terms of the a direct cross-linking reaction between the CRE and AuNPs, which physical implementation of the instrumentation in a portable and easy- causes an aggregation that produces an improved change of colour. to-handle device, rapid data acquisition and interpretation, analytical Despite the advantage of suppressing the use of picric acid, urine performance and portability. As continued monitoring of CRE levels is samples were diluted 100-fold because of the very low working range at not strictly necessary from a clinical point of view (Kasiske et al., 2001; the nM level. Sittiwong and Unob (2015) proposed a similar urinary Schmidt et al., 2017), long-term performance of the sensor is not re- CRE detection with AuNPs and CRE extraction on silica gel functiona- quired, and consequently, disposability of the sensor is a suitable al- lized with sulfonic acid. The aggregation of CRE with AuNPs promotes a ternative. This is an advantage once encountering possible (bio)fouling colour change that can be observed by the naked eye as well as by effects, which are more dramatic over time(Wang et al., 2018), and re- spectrophotometry. As in the previous case, the urine samples required calibration of the sensors to correct response drift (see a deeper dis- a previous dilution step before performing the measurements. Un- cussion in Section 4). fortunately, none of these examples shows values related to the accu- Accordingly, the main analytical features to be considered are: re- racy of the sensors against standard methodologies, although the time sponse time, selectivity and linear range. The ideal response time is required to perform the assays seems promising in most of them (be- within seconds-minutes, selectivity should allow for accurate CRE de- tween 1 and 10 min). tection in the biological fluid of interest – especially in blood –witha A slightly different proof-of-concept was reported by Du and co- sufficient limit of detection (LOD) and, finally, the linear range mustbe workers (Du et al., 2015). In this case, the study was based on the sy- as large as possible to include both healthy and harmful levels in order nergistic coordination chemistry of CRE and uric acid in the presence of to avoid reaching ‘false positives’ and/or ‘false negatives’. Notably, Hg2+ on AuNPs’ surfaces. Therefore, the colour of the solution changes healthy CRE levels in urine, which are in the mM range, are from 10- to from red to blue with rising CRE concentrations. The authors claimed 1000-fold higher than in the rest of the biological fluids (Table 1), the possibility of incorporating a smartphone as an on-field detector hence the same analytical technique would not be theoretically ap- and also for on-line data analysis. This work shows recoveries close to plicable to all samples. For one, urine samples are previously diluted, 100% (94–103%) for spiked CRE with an estimated analysis time of which is not compatible with the POC criteria. As a result, sample se- 5 min. In this regard, the POC potential of this approach may be lection is also analytically important, apart from attempting to reduce highlighted when overcoming the sample dilution requirement. any kind of invasion of the individual during sampling. Debus et al. (2015) reported certain technical innovations to de- Fig. 2 shows the percentage distributions of both the type of samples velop the Jaffé reaction in an improved setup close to the POC concept. and methodologies used in the publications listed in Table 2. As ob- Custom-made platforms were used for this purpose based on circular served, serum, plasma and urine are the only samples utilized for CRE dichroism spectroscopes and a computer screen photo-assisted tech- analysis. Worth noting is that in the majority of cases, a previous nique. CRE analysis in diluted urine needs between 5 and 15 min and sample dilution is required to be bracketed in the working range of the demonstrated accuracy deviations of ca. 10% (correlation of R2 = analytical methodology. Besides, the analysis of just artificial samples 0.930 – 0.977 with the Jaffé method), which falls inside traditional certainly appears with a high percentage at ca. 20% (Fig. 2a). Undiluted clinical tolerance limits. It is noteworthy that, as stated earlier, vali- whole blood and CSF are only utilized in two specific papers, likely dations employing the Jaffé method as gold standard and usually dis- owing to the matrix complexity as well as the complex and risky CSF play differences at this level (Walsh and Dempsey, 2002). Along the extraction. With respect to the techniques, colorimetric and electro- same lines, Fu and co-workers recently implemented the Jaffé reaction chemical methods continue exhibiting a clear dominance, ca. 38% of in a paper-based platform (microfluidic paper-based analytical device, the total number of papers in both cases (Fig. 2b). The interest in vol- µPAD) pursuing POC integration (Fu et al., 2018; Tseng et al., 2018). tammetry (cyclic voltammetry, CV, and differential pulse voltammetry, The analytical device serves as a CRE detector for blood and features a DPV) and amperometry has been growing over the years while only one reaction zone, which is placed after the serum separation from the potentiometric sensor has been published so far between 2014 and blood sample with picric acid and NaOH. When the blood diffuses 2018. In the following, we critically describe the advantages and dis- through the paper-based platform, the serum is separated in the fluidic advantages of these techniques. channel in a capillary manner (Yetisen et al., 2013). The µPAD is, in turn, inserted in a portable detection box that comprises a temperature 3.1. Colorimetric detection controller, power source, light source, CMOS camera (active pixel sensors in complementary metal-oxide-semiconductor) and smartphone In view of improving colorimetric sensing of CRE, some authors (connected to the system by USB or WiFi) to collect data in order to have recently reported variations of the Jaffé method (Table 2). Parmar build up the POC device (Fig. 3a). The linear range achieved with the et al. (2016) described a new approach using silver nanoparticles colorimetric detection in this device is wide enough to distinguish be- (AgNPs) coated with picric acid to generate the red chromogen. The tween healthy and harmful concentrations of CRE in blood/serum. combination of the NPs dispersion together with a centrifugation step These sensors required between 5 and 8 min to complete the assay and increases the efficiency of the reaction, which leads to a higher se- showed a good correlation with the gold standard technique (R2 = lectivity. Despite this approach being successfully applied to detect CRE 0.992 – 0.994). Importantly, this is one of the two (bio)sensors that in diluted CSF and serum, it seems not to be the best option for a POC have been truly applied for POC detection of CRE, in this case within implementation as these two biological fluids require a rather invasive whole undiluted blood. extraction and matrix separation, respectively, apart from the cen- Another approach based on the µPAD configuration in combination trifugation requirement. Later on, Alula et al. (2018) covered AgNPs with the multienzyme cascade reaction (tri-enzyme pathway), in which with citrate to promote alkaline CRE aggregation, demonstrating the CRE is involved, was reported by Talalak et al. (2015): possibility of CRE analysis in diluted urine. The main disadvantage is

116 R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124

Fig. 2. Summary of the biomatrices and analytical techniques used in modern CRE (bio)sensing. a) Distribution of the samples analysed in the papers listed in Table 2. b) Distribution of the methodologies for CRE detection collected in Table 2.

B. 1 : Creatinine+ H O Creatine 2 generated from the H2O2 reaction) of the light provided by six green LEDs for the colorimetric readout. Of benefit, the use of this device B. 2 : Creatine+ H2 O Urea+ Sarcosine permits a reduction in the LOD (~60 µM), and therefore, it is applicable B. 3 : Sarcosine+ H2 O Formaldehyde+ Glycine + H2 O 2 to plasma characterization and could be further applied to diluted urine. The enzymes involved in this cascade are creatinine or creatinine Other reactions have also been used to generate a colourful com- amidohydrolase (CA, 1 EC 3.5.2.10) (Step B.1), creatinase or creatine pound with CRE. Ellairaja et al. (2018) documented an approach where amidinohydrolase (CI, EC 3.5.3.3) (Step B.2) and sarcosine oxidase (SO, a chalcone, i.e., (E)-3-(pyren-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2- EC 1.5.3.1) (Step B.3). It is worth noting that other papers seen in en-1-one (PTP), was used as a chemodosimeter to interact with and Table 2 also described monitoring the formed H2O2 colorimetrically selectively recognize CRE in serum. The PTP chalcone works both as an (using a side reaction to generate a coloured complex) (Dal Dosso et al., absorbance and fluorescence probe. However, it presents a strong pH 2017) as well as amperometrically (Kumar et al., 2017a). Importantly, influence, i.e., at low pH values, the probe displayed two majorab- the other intermediate products could be used for indirect CRE detec- sorption peaks (at 297 and 407 nm) that vary when increasing the tion. The sensor reported by Talalak et al. (2015) was a paper-based concentration of CRE (measurable with UV–Vis spectroscopy). While strip with different zones created to first deliver the enzymes andthen this also takes place in fluorescence mode, the PTP probe exhibits an the reagents necessary to derivatize the generated H2O2 to quinonimine improved stability (lifetime of five weeks). The time necessary to for the colorimetric readout. The reported working range allows for complete the optical assay using is 10 min with recoveries of 88 – 105% CRE detection only in urine with analysis time close to 10 min. Ad- for spiked CRE. The main advantage with this sensor is the surprisingly vantageously, the sensor reported by Dal Dosso et al. (2017) based on wide detection range (from 1 nM to 1.4 mM), whereas the need for pH the same multienzyme cascade reaction was applied in undiluted control is the main drawback. Although the sensor was applied ex- plasma analysis, being the proposed device was really close to true POC clusively to CRE detection in serum, it could be suitable for urine when operation (Fig. 3b). The system integrates a cartridge based on a self- taking into account this range. powered imbibing microfluidic pump by liquid encapsulation With another approach, liquid crystals (LCs) were utilized for in- (SIMPLE). This cartridge is composed by different polymeric layers to direct CRE detection (Verma et al., 2016). The concept is based on the build the sampling and detection zones. In addition, the bioassay re- principle whereby the surface of the LCs is doped with hexylbiphenyl-4- quirements are fulfilled owing to the filtration (and selection ofthe carboxylic acid (HBA) and a local pH change induces a drastic colour proper wavelength at 570 nm to follow the colour of the compound

Fig. 3. POC devices for CRE detection reported over the last five years. a) Tseng et al. (2018): Photograph and illustrations of main components of the smart detection device, showing in detail the composition of the detection box. Reproduced from (Tseng et al., 2018), Copyright (2018), with permission of Elsevier. b) Dal Dosso et al. (2017): Overview of the Creacard (microfluidic cartridge), colorimeter and integrated Creasensor. A) Fabrication of the Creacard comprising the SIMPLE unitand detection unit. The assembly sequence of the cartridge is presented with red arrows and numbers. B) Top view of the assembled and prefilled Creacard and side view of the detection unit (chamber height=508 µm). C) Expanded view of the main components of the colorimeter. D) Expanded view of the Creasensor with the colorimeter, an Arduino microcontroller, power banks and screen. E) Photograph of the final Creasensor device in use. Reproduced from(Dal Dosso et al., 2017), Copyright (2017), with permission of Elsevier. c) Schematic representation summarizing the work presented by Kumar et al. (2017b): Mechanism of Fe(II)-based electrochemical detection of CRE and POC device in test mode with the inserted disposable test strip in which the sample is deposited.

117 R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124 change in LCs from a bright to dark optical appearance. In the sensor, palladium (Lad et al., 2008). Essentially, after the addition of CRE, part + the local pH change occurs owing to the generation of NH4 ions that of the free Fe(III) binds CRE, thereby forming a stable complex. This result after enzymatic conversion of CRE. The system was only applied binding produces a decrease in available Fe(III), which is observable in to detecting CRE levels in artificial samples up to 50 µM (healthy le- terms of the reduction of the peak current observed at the voltammetric vels), but also at harmful levels (synthetic samples of 100, 150 and wave. This POC device does not require any pre-treatment of the 1000 µM CRE). The orientational ordering of the LCs was determined sample and provides a CRE concentration in about one minute. Another with polarizing optical microscopy (×50). The system may have po- advantage is that FeCl3 chemistry has no interference via albumin, and tential for further POC application if the optical microscopes are re- this is very useful regarding the albumin-to-CRE ratio detection. How- placed by a simpler colorimetric detector, such as a camera. Never- ever, the current limitation of this sensor is in relation to the linear theless, the demonstration of the suitability for real sample analysis is range that only includes healthy CRE levels. Moreover, the authors mandatory before this implementation. claimed further steps based on sensor evaluation regarding the long- Importantly, before the selected period that this review covers, term stability and reliability of the test strips as well as interference some authors investigated CRE recognition by hydrogen binding with studies with samples from patients at advanced stages of renal disease distinct neutral hosts and supramolecular receptors with possible UV (macroalbuminuria). recognition of the formed complex (Buhlmann and Simon, 1993; In other work, QDs were utilized for selective CRE recognition Buhlmann et al., 1998). However, none of them further evolved be- (Hooshmand and Es’haghi, 2017). However, the sensitivity of this ap- cause of the better performance of the Jaffé method. proach was limited by the amount of QDs, sample pH, equilibration time of the sample with the QDs (0, 10 or 15 s) and scan rate in the 3.2. Electrochemical techniques voltammetry readout. Regarding the latter, low scan rates are prefer- able (0.01 and 0.05 V s–1). The sensor displayed a wide working range, Voltammetric detection of CRE employing electrodes based on which permits CRE detection in urine and serum. In our opinion, the molecularly imprinted polymers (MIPs) was reported in three recent translation of this concept into a paper-based platform able to separate papers (Diouf et al., 2017; Rao et al., 2017; Wen et al., 2014). In plasma from blood prior to the QD-based detection could be considered principle, the cavity developed in the MIPs has the specific size and a further step towards a potential POC device. shape to selectively host the analyte of interest. Diouf et al. (2017) Desai et al. (2018) demonstrated the indirect detection of CRE in reported a MIP-based sensor where the binding sites for CRE are gen- spiked urine samples by CV and DPV analysis of cysteine. For this erated onto a carboxylic polyvinyl chloride layer firstly deposited on purpose, the cysteine protease enzyme, papain, was immobilized in a gold electrodes. This sensor selectively identifies CRE with a LOD atthe screen-printed electrode modified with carboxyl-functionalized multi- nM level (0.14 nM) and was successfully applied to detect CRE in di- walled carbon nanotubes. The amino group of papain is, in turn, luted urine. While the reported low LOD would allow for CRE detection covalently bound to the carboxyl group on the electrode surface. Then, in any biological fluid subsequent to dilution, there was a lack ofin- the interaction between papain and Cystatin C is electrochemically terference. This may limit application to whole blood or other samples. monitored, showing an extremely low LOD of 0.006 nM with a minimal Besides this, the long analysis time required for the entire assay (ca. 2 – requirement of sample volume (6 µL). The main disadvantage is that the 5 h) is a marked disadvantage. presented working range means any sort of sample has to be diluted The studies presented by Rao and Wen showed two similar ex- prior to analysis, thus inducing more error in the CRE detection and amples of MIPs based on the use of NPs, such as NiNPs or Fe3O4NPs, being somehow incompatible with the POC concept. together with polyaniline (PANI) (Rao et al., 2017; Wen et al., 2014). In Dasgupta et al. (2018) reported indirect CRE detection by ampero- both cases, glassy carbon electrodes are modified with the MIP and metric monitoring of Fe(III) concentration after its reaction with CRE, a nanoparticle-PANI composite by electropolymerization, yielding si- similar concept to that reported by Kumar et al. (2017b), but the in- milar LODs (0.2 and 0.35 nM respectively). The sensors were applied to teraction between CRE and FeCl3 occurs directly in solution, which detect CRE in spiked (artificial) samples, leading to recovery percen- takes analysis time of 10 min. Despite an appropriate working range tages close to 100%. In addition, one of the main disadvantages of using being presented, the methodology should to be heavily evaluated voltammetric sensors based on MIPs is the great amount of time (ca. considering real serum samples and interference studies. The other two 1–3 h) needed for preparation taking into account the washing, in- papers with amperometry readout comprise enzymatic reactions where cubation and polymerization steps. CRE is involved (see Table 2) displaying faster response times (few Other voltammetric sensors are based on the monitoring of CRE seconds). Zhybak et al. (2016) immobilized the required enzymes by following selective binding with Cu(II), Fe(III) or quantum dots (QDs) drop-casting a solution of CRE deiminase and urease enzymes onto a Cu (Raveendran et al., 2017; Kumar et al., 2017b; Hooshmand and electrode modified with PANI and Nafion. Conversely, Kumar et al. Es’haghi, 2017) as well as enzymatic conversion to cysteine (Desai (2017a) employed an immobilization procedure comprising the dip et al., 2018). Raveendran et al. (2017) proposed a sensor capable of coating of the electrode inside a solution that contains NPs of creati- detecting CRE by voltammetry, in particular monitoring the activity of ninase, creatinase and sarcosine oxidase enzymes. The NPs aggregate a copper-CRE complex. The sensor showed a wide linear range from 6 and attach onto the electrode surface after 2 h. In both cases, analytical to 378 µM (LOD = 0.07 µM) that allows distinguishing between healthy performance is apparently promising with respect to POC CRE detection and harmful concentrations as a result of CRE analysis in both serum (response time of 15 and 2 s, LOD of 0.5 and 0.01 µM, and robust sto- and diluted urine (recoveries 95 – 98% of spiked CRE). Nonetheless, the rage stability of three months without losing analytical performance as high selectivity of this sensor for CRE in serum was brought about by a well as eight months with a loss of only 10% of initial enzymatic ac- pre-incubation step of the sample with PbO2 powder, which ensured the tivity in Zhybak and Kumar studies, respectively). Indeed, these bio- rejection of interferences, such as uric acid during the detection. Note sensors have been successfully applied for CRE detection in (diluted) that all these steps made the approach incompatible with the POC serum. Both sensors exhibited a robust correlation compared with the concept, apart from the really long analysis time (more than 30 min). standard spectrophotometric method during their validation. The sensor presented by Kumar et al. (2017b) was based on a Another example based on electrochemical detection comprises an carbon-printed electrode coated with a FeCl3 cotton fibre membrane ion-sensitive field effect transistor (ISFET) in Ozansoy et al. (2017). In with the ability to measure CRE in urine samples without previous di- this investigation, the immobilization of the CD enzyme was accom- lution (Fig. 3c). Remarkably, this is the only voltammetry sensor that plished on zeolite-based electrodes using glutaraldehyde. For this pur- displayed major success as a POC CRE detector. Detection was based on pose, the surface of the sensor was firstly modified by drop-coating four CRE forming complexes with metals, such as platinum, copper, iron or different zeolites together with the enzyme. These zeolite-based sensors

118 R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124 all displayed improved analytical parameters for CRE detection with et al., 2018) and electrochemiluminescence (ECL) (Babamiri et al., sensitivities between 21 and 30 µA/mM, LOD of 5 µM and a response 2018), the latter displaying remarkably broad working ranges. Hanif time of 120 s. The sensors were only applied to CRE detection in arti- et al. presented enhanced CL measurement of the reaction between CRE ficial samples. and hydrogen peroxide in the presence of cobalt ions. The CRE de- CRE detection has been additionally accomplished by exceptionally termination in diluted urine samples was accomplished by coupling CL simple electrochemical techniques, such as potentiometry and con- detection to flow-injection analysis, which involves a really sort ana- ductimetry. Indeed, from the ammonium detection introduced by lysis time of some minutes. Despite the technique's simplicity, the pH Meyerhoff and Rechnitz (1976), other authors have described po- influence is relatively marked (i.e., CL intensity rises with higherpH tentiometric biosensors with enhanced analytical performances, in- because it facilitates the deprotonation of H2O2 and oxidation of CRE as cluding the integration of a chitosan membrane with the immobilized well as the solubility of cobalt ions decreasing in the medium). enzyme and implementation of the ammonium-selective electrode in Babu et al. (2018) reported a PL approach based on carbon nanodots flow-injection analysis (Radomska et al., 2004a; Magalhaes and (CNDs) on carbon-gold nanocomposites (C-Au NCs) functionalized with Machado, 2002). The conversion of CRE into a cation that is measur- bovine serum albumin (BSA). The intensity of PL diminishes upon in- able potentiometrically was always necessary based on the absence of creasing concentration of CRE because of the removal of BSA from the an effective receptor (known as an ionophore in the jargon) to directly composite after reaction with CRE. The sensor displays an extremely sense CRE (or its cation, creatininium, at pH values below 3.8). broad working range (17 µM–1.7 M) where healthy and harmful levels To this end, Ballester and colleagues reported an effective iono- of CRE in all undiluted biological fluids are included. The work also phore-based potentiometric sensor for creatininium (Guinovart et al., provided a deep biocompatibility study producing promising results in 2016, 2017), a calix[4]pyrrole-based molecule that offers a suitable comparison with other fluorescent dyes and QDs. However, there isa selectivity for creatininium considering the main interfering ions in lack of demonstration of the sensor in real samples. Babamiri et al. biological fluids: logKurea, CRE = 4.3; logKCa, CRE = 4.8; logKNa, CRE = (2018) reported the determination of CRE using ITO-MIPs electrodes

3.7; logKK, CRE = 2.5; logKNH4, CRE = 2.3; logKcreatine, CRE = 3.5. De- modified with nickel nanoclusters (NiNCs) as ECL emitters. TheECL spite the potentiometric sensor claimed to be an all-solid-state type, in signal is produced when the electrode reacts with tri-n-propylamine fact, it belongs to the well-known coated wire electrode (CWE) under- after CRE insertion in the corresponding MIP cavities, which is a pro- scored by Cattrall et al. (1974). This is in part because the assembled cess that takes a longer time (15 – 30 min) than other concepts for CRE sensor does not incorporate any ion-to-electron transducer, which un- recognition. Unfortunately, the results were validated by spiking the doubtedly negatively impacts stability and drift of the potentiometric samples with known CRE amounts and calculating recoveries, which is response. However, the authors reported adequate analytical perfor- not the most convenient validation protocol. mance, such as wide linear range embracing healthy and harmful CRE Stamping surface-enhanced Raman scattering (S-SERS) was recently levels in diluted urine and plasma. In addition, the sensor was sa- utilized for CRE detection in artificial samples (Li et al., 2015). The tisfactorily validated with the gold standard Jaffé method. The main study showed the combination of the S-SERS technique with nano- disadvantages are the need for buffering the sample at pH 3.8to porous gold disk (NPGD) plasmonic substrates. It was shown that the transform all the neutral CRE into the creatininium cation together with CRE peak intensity rises as a function of CRE concentration owing to the the mandatory necessity of diluting the sample by factors of 100 and 10 interaction with the NPs. S-SERS was claimed here as an excellent for urine and plasma, respectively, to minimize biofouling contributions method to obtain a CRE spectrum at 100 nM concentration. In addition, to the electrode response. Despite these drawbacks keeping this sensor a study based on surface plasmon resonance (SPR) was recently pub- away from POC application, it seemingly demonstrated potentially fa- lished (Pothipor et al., 2017) where the transmission surface plasmon vourable results detecting CRE in diluted samples from both patients resonance-imaging (TSPR-i) was investigated on a gold substrate, which that are healthy and those suffering from renal dysfunction (Guinovart was modified with AgNPs by means of a microfluidic cell. TheTSPR-i et al., 2016). intensity first decreased after the deposition of the AgNPs and thenwas Finally, Braiek et al. (2018) presented conductometric detection of raised after CRE incubation for 1 h because the strong CRE-AgNPs in- CRE by means of the immobilization of the CD enzyme onto a com- teraction. The displayed working range at the mM levels restricts the posite film (composed of polyvinylalcohol/polyethyleneimine/gold application of this principle to urine, but, unfortunately, the authors of nanoparticles, PVA/PEI/AuNPs). The final conductometric biosensor both works only provided evidence for CRE detection in artificial showed robust reproducibility along with broad linear range and was samples (see Table 2). used for CRE detection in spiked artificial serum samples (estimated Solid phase microextraction (SPME) coupled to ultrafast evapora- time of 3 min). Validation with real samples and correlation via stan- tion with ion mobility spectrometry (IMS) readout has been proposed dard methods were not addressed in this study. by Jafari et al. (2015). The approach was validated with the Jaffé method in two serum and urine samples showing recovery values close 3.3. Other methods to 100%. Both samples required a 1:1 dilution for the analysis in order to fit within the working range. The very low sample volume required CRE detection based on fluorescence was reported in two different for the analysis (10 µL) can be considered a remarkable advantage. works by the groups of Ellairaja (already discussed in colorimetric Nevertheless, in order to be more consistent with respect to reliability methods) and Pal (Ellairaja et al., 2018; Pal et al., 2016). In the latter of this new approach, validation with a higher number of real samples work, a Pd2+-naphthalimide-based florescence light-up probe (FCP-Pd) from healthy and CKD patients should be performed additionally, as put was developed to detect CRE in serum samples as low as 30 µM. The forth by the authors. detection was based on the fluorescence recovery of FCP by removing Pd2+ (the well-known heavy atom-quenching effect) from the FCP-Pd 4. General criticism of the most promising principles for complex after an incubation step of 30 min in the presence of CRE. creatinine (bio)sensing at the point-of-care level During this time, CRE reacts with Pd2+ to form a complex (Pd (CRE)2Cl2), therefore providing free FCP ligand. The approach was Having described all the techniques reported for CRE detection over validated with the colorimetric Jaffé method, showing recoveries of the last five years, the following conclusions can be extracted con- 95–99% for spiked CRE, therefore indicating the suitability of this new sidering POC application of all these (bio)sensors: system for further clinical applications. Luminescence approaches were also reported, including chemilu- (i) Depending on the working range of the (bio)sensor, this will be minescence (CL) (Hanif et al., 2016), photoluminescence (PL) (Babu suitable for various kinds of biological fluids. Considering urine, a

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range from 1 to 30 mM will allow for the identification of healthy papers can be considered truly biosensors, i.e. the sensor uses a and harmful CRE levels, while in the case of blood (or plasma/ recognition element of biological nature (Perumal and Hashim, serum), this range is from 10 to more than 150 µM (see Table 1). 2014), and only the colorimetric sensor developed by Dal Dosso The latter would be also suitable for the remaining biological et al. has been applied for POC CRE detection. In this context, CRE fluids. Of note, these estimated ranges would permit one topre- detection is achieved through an enzymatic reaction and the re- cisely quantify CRE levels at healthy levels and qualitatively quired enzyme(s) are traditionally immobilized in any of the parts identify outlier cases. of the device. Advantageously, immobilized enzymes are more (ii) While working ranges at the mM level restrict the use of the (bio) resistant to external changes and permits to develop the enzymatic sensors for urine, µM levels permit CRE detection in blood and reaction in a controlled and confined environment. Indeed, CRE diluted urine. However, in the specific cases where nM levels or biosensors collected in Table 2 present long-lasting lifetime as well even lower are achieved, dilution is required for all samples (Du as the faster analysis times (between seconds and few minutes). et al., 2015; Desai et al., 2018; Rao et al., 2017; Diouf et al., 2017). Apart from dip-coating and drop-casting approaches reported by Unfortunately, dilution steps impede the POC application of the Kumar et al. (2017a) and Talalak et al. (2015), there is a very (bio)sensor. broad catalogue of strategies for general enzyme immobilization (iii) Detection methods that do not imply colorimetric or electro- (Cosnier, 1999; Datta et al., 2013) that may be used in further chemical readouts generally involve more sophisticated sensing designs for POC CRE detection (i.e., improving lifetime, enzyme principles and larger instrumentation. Within this context, fluor- activity and, indeed, putting forth physical/chemical barriers to escence and luminescence probes seem to be more suitable for POC interference (Yan et al., 2011)). While, all these techniques are in CRE detection in the near future (Ellairaja et al., 2018; Pal et al., principle easy to be implemented with any type of enzyme, the 2016; Babamiri et al., 2018; Babu et al., 2018; Hanif et al., 2016). complexity of the procedure increases in the case of cascade re- However, data interpretation is also more complex than with col- actions involved in the biosensing process. Moreover, it is abso- orimetric and electrochemical options considering that the data lutely necessary to assure the correct performance of all the en- will be handled and interpreted by non-expert end users. In our zymes under optimal pH and temperature, which sometimes limits opinion, this is the reason why colorimetric and electrochemical the use of the biosensor or involves a change in the pH of the CRE detection is more spread. sample. (iv) Apart from the attempts to translating the Jaffé method at the POC Apart from an increase in the cost of the sensor manufacturing, the level (Fu et al., 2018; Tseng et al., 2018), only one colorimetric involvement of multiple enzymatic reactions additionally increases sensor and other voltammetric were successfully applied for this the time required for the analysis, which will be here based on the endeavour (Kumar et al., 2017b; Dal Dosso et al., 2017; see Fig. 3). needed time for the sample to reach the reagents and compounds Significantly, the only path to establish the traditional Jaffé reac- in the device, the reaction times associated with the enzymatic tion for CRE POC detection in blood is using a paper-based plat- reaction and the generation of coloured products (if necessary) form that allows for the in situ separation of plasma from the blood together with the response time of the biosensor. For instance, in sample in the same device without the need to transport the the case of the tri-enzyme reaction, this time is close to 5 or 7 min sample to a centralized laboratory. Herein, it is absolutely neces- depending on the approach employed to detect H2O2 (Kumar et al., sary to investigate how the physical separation process itself spe- 2017a; Dal Dosso et al., 2017). Beneficially, other reactions based cifically influences CRE as a neutral biomolecule. on synthetic enzymes may be also explored in the close future The biosensor reported by Dal Dosso et al. (2017) is based on the seeking to overcome all drawbacks of current biosensors.

CRE enzymatic reaction to produce H2O2, which is later derived Other advantage involved in the use of biosensors for CRE detec- into a coloured compound. The monitoring of one of the possible tion is the possibility of regeneration of the analytical signal. Al- products when CRE is involved in enzymatic reactions is a widely though the disposability of the sensor undoubtedly fits with the used strategy for CRE detection in both blood and urine (see POC concept, if its cost does not permit this way of operativity, Table 2). Indeed, the majority of the commercially available de- regeneration of the sensing part is the best option. The selected vices claiming POC CRE detection are based on an enzymatic re- process for this purpose may differ according to the nature of the action coupled to amperometric or spectrophotometric detection sensing/recognition element. According to Goode et al., in general, (Shephard, 2011). Despite these devices being suitable for use in there are five types of mechanisms for the regeneration of bio- clinical laboratories after sample collection, none of them really sensors: (i) chemical regeneration to alter the solvent environment fulfil the current requirements for a POC system (Mahato et al., and favour the unbinding of the target; (ii) thermal regeneration 2017; St John and Price, 2014), mainly because they are cum- that gives molecules an increased kinetic energy allowing binding bersome to measure next to the patient. Importantly, there are two forces; (iii) regeneration promoted by enthalpic interactions devices (called as iSTAT and Stat Sensor)4 with true POC potential (mainly related to polar or ionic charges and potential energy in- in terms of dimensions, blood detection, analysis time (2 and volved in chemical bonds); (iv) through entropic interactions (in 0.5 min, respectively), sample volume (65 and 1 µL, respectively) case of hydrophobic properties), and finally, (v) electrochemical and working range, including healthy and harmful levels regeneration by applying negative potentials that provide highly (18–1768 µM and 27−1056 µM, respectively), but there are still controlled and localized processes (Goode et al., 2015). improvements necessary regarding selectivity. Other examples of (vi) The device reported by Kumar et al. (2017b) may awaken interest commercial devices are Roche-Reflotron Plus and Abaxis-Piccolo of the reader for different reasons (Fig. 3c). First, it is not a bio- that offer excellent analytical features in terms of analysis time(3 sensor per se because it does not comprise the use of any enzyme and 8.5 min, respectively), sample volume (30 and 9 µL, respec- (biological component of the biosensor). Instead, it is based on the tively) and working range (45–884 µM and 18–1768 µM, respec- CRE interaction with Fe(III). Second, it is the only POC device that tively), but they involve the use of large and heavy instrumenta- measures CRE in a fast, reliable and disposable manner in urine. tion (5 kg in both cases), which limits its use as POC devices. Last but not least, the sensing principle is very simple, and the (v) Among all the sensors collected in this review, only 7 of the 33 observed signal is easy to interpret with results that can be validated. It is worth highlighting that according to Table 2, there is an emerging trend to utilize reactions involving CRE rather than en- 4 https://www.pointofcare.abbott/us/en/offerings/istat/istat-test-cartridges; zymatic ones. Importantly, there are many different reactions that http://www.novabio.us/statstrip-creatinine/ have been explored, but in our opinion, efforts to show the true

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potential for POC CRE detection is still necessary, for example, stage 3, showing acceptable correlation with the Jaffé method (0.85 – binding with adenosine, citrate, chalcone or AuNPs in the presence 0.95) (Kumar et al., 2018). The disposability nature of the sensors based of NPs (Du et al., 2016; Alula et al., 2018; Ellairaja et al., 2018; He on traditional strips facilities the use in such large number of samples. et al., 2015) or with Pd (Pal et al., 2016), H2O2 (Hanif et al., 2016), The colorimetric biosensor reported by Dal Dosso et al. (2017) is not Cu(II) (Raveendran et al., 2017) and QDs (Hooshmand and truly a POC device because it is not able to measure directly in blood as Es'haghi, 2017) among others. serum isolation is mandatory. As a consequence, although the authors stated that this is a POC device (Fig. 3b), we would like to put forth we In general, any POC detection involves the following analytical re- believe it is a close-to-POC device. Through further steps, the authors quirements for the sensing principle (Junker et al., 2010; Mahfouz may resolve the issue of application in whole blood using a paper-based et al., 2013; Zarei, 2017): fast response time (from seconds to minutes); device. Furthermore, a more exhaustive investigation of the standar- reliability (clinical tolerance limits generally require 10 – 15% accuracy dization of the calibration graph, the lifetime of the sensor (and en- in the validation protocol); adequate selectivity to afford the clinically zymes) as well as a more elaborated validation with a higher number of demanded working range and LOD; no or minimum risk of sample real samples will be necessary in the near future while also identifying contamination (which is incompatible with any sample manual hand- harmful clinical situations. In addition, the fabrication and handling of ling, such as dilution, plasma extraction in blood, mixture of reagents the device seems to be complex (Fig. 3b). Interestingly, considering and others); disposability to avoid any risk of cross-contamination; easy lifetime studies of biosensors, long-term investigations of the activity of or null calibration protocol; preparation of the sensing principle must the enzymes were conducted during more than three and eight months be compatible with mass production and, in the case of requirements, (Zhybak et al., 2016; Kumar et al., 2017a). In this regard, some authors only easy steps should be carried out by the end-user because the re- claim that the remarkable robustness and stability of immobilized en- liability of the quantification will be influenced by all these steps; tol- zymes become from the structural changes produced during the im- erable provision of ‘false positives’ and ‘false negatives’; among others. mobilization procedure that generates a new microenvironment (totally Considering the three (bio)sensors for CRE detection found in different from the bulk solution) where the enzyme acts in amore publications of the last five years and of which the authors remark they controlled manner (Homaei et al., 2013). developed POC devices, it is certain they are not. None of them really Lastly, the colorimetric sensor developed by Tseng et al. (2018) still fulfil all the POC requirements, although they are very close(Dal Dosso features the majority of the drawbacks innate to the Jaffé reaction et al., 2017; Kumar et al., 2017b; Tseng et al., 2018). The amperometric (already mentioned in the previous sections), though it resolves blood biosensor developed by Kumar et al. (2017b) is based on the interaction utilization (Fig. 3a). However, in our opinion, it is necessary to reach a between Fe(III) immobilized on a strip and CRE in the sample. It pre- paradigm shift from the Jaffé reaction to another different strategy that sents a LOD in the mM range and it is therefore applicable only to urine provides authentic CRE detection fulfilling all POC requirements. analysis without the need for any pre-treatment. The device is already Consequently, we are now in the position to answer the question that implemented in a friendly interface as to how the disposable strip is was raised in the abstract of the present review: To what extent is placed, and in just one min, the CRE content in 300 µL of urine is ob- creatinine detection already solved in healthcare applications, or is still tained (Fig. 3c). The device was validated in a first step with 50 urine a challenge? The response is straightforward. While CRE detection is samples and the results were well-correlated with those obtained possible at the laboratory scale via the Jaffé method and others through the Jaffé method. There is no temperature influence inthe (Shephard, 2011), there is not yet one biosensor for true POC detection range of 20–40 °C, and no interference from albumin, though other of CRE that allows for trustworthy clinical decision-making. As there is interferences were not tested. Regrettably, while the calibration graph an urgent need to deal this challenge, novel CRE-sensing concepts have is considered a universal one and therefore implemented into the in- been proposed over the last five years and are positively evolving to- terface and software to calculate CRE concentration, there is no ex- wards POC CRE detection. haustive investigation of the appropriateness of this standardization Within this context, the ideal device may probably use one single with a statistical approach. It would be necessary to indicate the lim- blood drop (fingerprint technique), or low urine volume (after 24h itation in accuracy and precision of the quantitative detection as well as collection), or indeed could be conceived as a wearable sensor em- establishing the re-calibration frequency of the sensor. Indeed, funda- bedded in a diaper or urine collection bag, or even implemented in a mentally speaking, amperometric sensors always need re-calibration microneedle-based platform for advanced CRE detection in ISF. In these except in the case that linearity is found between the analyte con- latter cases, additional biocompatibility and toxicology studies related centration and the involved charge (i.e. coulometric sensors; Cuartero to the skin are necessary for a final prototype. et al., 2015). On the other hand, efforts carried out in the laboratory surrounding In addition, the preparation of the strip necessitates several steps - the investigation of (bio)sensor performance must be reformulated in deposition of FeCl3 solution in cotton substrate that is then placed on order to consider mandatory information for the POC concept that go the plastic strip with the PINE electrode and dried at 34 °C in an oven. unnoticed in recent publications. We are referring to: (i) lifetime eva- Furthermore,there is no specification surrounding the stability of the luation of the (bio)sensor (and required materials) to establish suit- electrodes, i.e., if this is an approach compatible with mass production ability for further mass production and easy utilization by non-expert or if some of the preparation steps need to be executed by the end user. end users; (ii) investigation of any (bio)fouling effect (Wang et al., It could also be necessary to further demonstrate the utility of the 2018) during the time in which the sensor is in contact with the sample sensor for the identification of harmful clinical situations. Despite these (especially in the case of blood); (iii) characterization of the dis- uncertainties, it is clear that the device proposed by Kumar et al. posability feature, standardization of the calibration graph and re-ca- (2017b) is very close to POC CRE detection (Fig. 3c). In a very recent libration frequency by using one- or two-point protocols, as in the work, the authors presented the application of the POC device for early glucometer, pH meter or recently established environmental sensors detection of diabetes kidney disease (DKD) (Kumar et al., 2017b). The (Cuartero et al., 2017); (iv) deeper interference study considering ma- device is feasible to quantitatively measure five different biomarkers trix effects and avoiding the need for sample dilution; (v) clinical va- related to DKD and Diabetes mellitus among which urine CRE and the lidation based on gold standard techniques (Jaffé method, HPLC or any ACR ratio are presented. This study represents a major step forward in other commercially available device) and using a significantly higher realizing a robust and scalable POC multianalyte sensor able to gen- number of samples (> 50 for prototypes during early-stage develop- erate clinically relevant observations in less than one minute. The va- ment), including healthy and harmful levels; and (vi) technical re- lidation of this patented device was accomplished using more than 500 quirements, such as cost-effective fabrication, self-powered devices, samples from 400 patients suffering DKD at stage 2 and 30 patients at data harvesting and (wireless) connections, need to be additionally

121 R. Cánovas et al. Biosensors and Bioelectronics 130 (2019) 110–124 considered, albeit this is outside the scope of the present review. concentrations in uraemic patients. Urol. Res. 25, 337–340. de Almeida, P.D.V., Grégio, A.M.T., Machado, M.Â.N., de Lima, A.A.S., Azevedo, L.R. Saliva, 2008. Saliva composition and functions: J. Comte. Pract. 9, 72–80. 5. Conclusion Alula, M.T., Karamchand, L., Hendricks, N.R., Blackburn, J.M., 2018. Citrate-capped silver nanoparticles as a probe for sensitive and selective colorimetric and spectro- The clinical importance of detecting CRE levels in urine and espe- photometric sensing of creatinine in human urine. Anal. Chim. Acta 1007, 40–49. Assadi, F., 2008. Diagnosis of hypokalemia: a problem-solving approach to clinical cases. cially in blood (serum) has been extensively supported by alliances Iran. J. Kidney Dis. 2, 115–122. between chemists and clinicians. Modern creatinine (bio)sensing is Babamiri, B., Salimi, A., Hallaj, R., Hasanzadeh, M., 2018. Nickel nanoclusters as a novel based on distinct approaches, among them involving the Jaffé reaction, emitter for molecularly imprinted electrochemiluminescence based sensor toward multienzyme cascades or specific creatinine binding. The two common nanomolar detection of creatinine. Biosens. Bioelectron. 107, 272–279. Babu, P.J., Raichur, A.M., Doble, M., 2018. Synthesis and characterization of bio- detection methods used are colorimetry (VIS absorption) and electro- compatible carbon-gold (C-Au) nanocomposites and their biomedical applications as chemistry (including voltammetry and amperometry) though fluores- an optical sensor for creatinine detection and cellular imaging. Sens. Actuators, B cence and luminescence detection that have recently showed promising Chem. 258, 1267–1278. Bagalad, B., Mohankumar, K., Madhushankari, G., Donoghue, M., Kuberappa, P., 2017. features for creatinine detection. The majority of the works reported Diagnostic accuracy of salivary creatinine, urea, and potassium levels to assess dia- over the last five years are applied to urine or plasma/serum, while only lysis need in renal failure patients. Dent. Res. J. (Isfahan). 14, 13–18. one sensor is suitable for undiluted whole blood detection (Tseng et al., Bandodkar, A.J., Jeerapan, I., Wang, J., 2016. Wearable chemical sensors: present challenges and future prospects. ACS Sens. 1, 464–482. 2018) and another demonstrating to be appropriate for cerebral spinal Bartz, S.K., Caldas, M.C., Tomsa, A., Krishnamurthy, R., Bacha, F., 2015. Urine albumin- fluid (Parmar et al., 2016). Of all the inspected (bio)sensors, only three to-creatinine ratio: a marker of early endothelial dysfunction in youth. J. Clin. are claimed –by the authors– to be for creatinine detection at the POC Endocrinol. Metab. 100, 3393–3399. Bass, D.E., Dobalian, I.T., 1953. Ratio Between true and apparent creatinine in sweat. Am. level (Dal Dosso et al., 2017; Kumar et al., 2017b; Tseng et al., 2018). Physiol. Soc. 5, 555–558. Nevertheless, we believe that these (bio)sensors need further im- Baxmann, A.C., Ahmed, M.S., Marques, N.C., Menon, V.B., Pereira, A.B., Kirsztajn, G.M., provements and much more intensive evaluations towards true POC Heilberg, I.P., 2008. Influence of muscle mass and physical activity on serum and urinary creatinine and serum cystatin C. Clin. J. Am. Soc. Nephrol. 3, 348–354. applications in healthcare. In contrast, clinical decision-making invol- Boeniger, M.F., Lowry, L.K., Rosenberg, J., 1993. Interpretation of urine results used to ving CRE levels currently rely on the Jaffé method and, to a lesser ex- assess chemical exposure with emphasis on creatinine adjustments: a review. Am. tent, on other commercial devices based on the amperometric mon- Ind. Hyg. Assoc. J. 54, 615–627. itoring of CRE enzymatic reaction products. As a result, the analysis of Braiek, M., Djebbi, M.A., Chateaux, J.F., Bonhomme, A., Vargiolu, R., Bessueille, F., Jaffrezic-Renault, N., 2018. A conductometric creatinine biosensor prepared through CRE is yet to be centralized at clinical laboratories, impeding data ac- contact printing of polyvinyl alcohol/polyethyleneimine based enzymatic membrane. quisition in real time and therefore delaying the associated medical Microelectron. Eng. 187–188, 43–49. action. Certain key facts highlighted herein may contribute to the es- Buhlmann, P., Amemiya, S., Nishizawa, S., Xiao, K.P., Umezawa, Y., 1998. Hydrogen- bonding Ionophores for Inorganic anions and nucleotides and their application in tablishment of the new routes towards a definitive solution for CRE chemical sensors. J. Inc. Phenomen. Mol. Recog. Chem. 32, 151–163. discernment in healthcare applications. Experts in the field have Buhlmann, P., Simon, W., 1993. Neutral hosts for the complexation of creatinine. pointed out the associated drawbacks using the Jaffé method and be- Tetrahedron 49, 7627–7636. Cattrall, R.W., Tribuzio, S., Freiser, H., 1974. Potassium ion responsive coated wire cause of that, new sensing principles have been actively proposed. electrode based on valinomycin. Anal. Chem. 46, 2223–2224. Importantly, other biological fluids with great potential for CRE de- Cherney, D.Z.I., Zinman, B., Inzucchi, S.E., Koitka-Weber, A., Mattheus, M., von Eynatten, tection are not yet fully explored. This is the case for saliva and ISF. To M., Wanner, C., 2017. Effects of empagliflozin on the urinary albumin-to-creatinine ratio in patients with type 2 diabetes and established cardiovascular disease: an ex- date, none of the reported strategies are suitable for the direct detection ploratory analysis from the EMPA-REG OUTCOME randomised, placebo-controlled of CRE in whole blood, and the only device that uses blood for the trial. Lancet Diabetes Endocrinol. 5, 610–621. analysis includes the previous plasma separation by means of capillaries Cho, Y.H., Craig, M.E., Davis, E.A., Cotterill, A.M., Couper, J.J., Cameron, F.J., Benitez- Aguirre, P.Z., Dalton, R.N., Dunger, D.B., Jones, T.W., Donaghue, K.C., 2015. Cardiac via a paper-based platform. Consequently, it seems that a POC device autonomic dysfunction is associated with high-risk albumin-to-creatinine ratio in based on the fingerprint technique is not accessible with modern CRE young adolescents with type 1 diabetes in adDIT (Adolescent Type 1 diabetes cardio- (bio)sensing. Finally, there are a number of weaknesses in the way that renal interventional trial). Diabetes Care 38, 676–681. the (bio)sensors are characterized, mainly related to calibration, po- Cockcroft, D.W., Gault, M.H., 1976. Prediciton of creatinine clearance from serum creatinine. 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In Situ Detection of Species Relevant to the Carbon Cycle in The authors acknowledge the financial support of KTH Royal Seawater with Submersible Potentiometric Probes. Environ. Sci. Technol. Lett. 4, 410–415. Institute of Technology (Starting Grant Programme, K-2017-0371), Dal Dosso, F., Decrop, D., Pérez-Ruiz, E., Daems, D., Agten, H., Al-Ghezi, O., Bollen, O., Swedish Research Council (Project Grant VR-2017–4887), Wenner- Breukers, J., De Rop, F., Katsafadou, M., Lepoudre, J., Lyu, L., Piron, P., Saesen, R., Gren Foundation (Scholarship UPD2017-0220) and the European Union Sels, S., Soenen, R., Staljanssens, E., Taraporewalla, J., Kokalj, T., Spasic, D., Lammertyn, J., 2017. Creasensor: simple technology for creatinine detection in (Marie Skłodowska-Curie Individual Fellowship European, H2020- plasma. Anal. Chim. Acta 1000, 191–198. MSCA-IF-2017, Grant no. 792824). R.C thanks the Alfonso Martin Dasgupta, P., Vinay Kumar, B., Krishnaswamy, R., Navakanta Bhat, B, B.P., 2018. 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